Alternative splicing plays a crucial role in the life cycle of the DNA tumor virus SV40, as well as other viruses, and in the expression of numerous cellular genes. For example, the SV40 early transcript can be spliced to produce the mRNAs encoding both the large tumor antigen, which is required for both viral replication and oncogenic cell transformation, and the small tumor antigen, whose function is also related to cell growth. Despite significant advances in our understanding of the general mechanism of pre mRNA splicing, little is known about the factors that control alternative splicing. This question is of particular significance because of the increasing evidence that regulated splicing plays an important role not only in the replication of oncogenic viruses, but also in the control of genes that are expressed at different developmental stages or in different tissues. The experiments proposed here are designed to provide insights into the factors that control alternative splicing, how they function and how they themselves are controlled. The following specific issues will be addressed: Using in vitro mutagenesis techniques, sequences crucial for controlled alternative splicing of SV40 pre mRNAs will be identified both by transfection of different types of cultured mammalian cells, and by micro-injection of X. laevis oocytes. The pathways and parameters of SV40 early splicing will be investigated by using extracts of mammalian cells to process pre mRNAs in vitro. Also using in vitro processing systems, the identities of trans-acting factors that influence the choice of alternative splicing pathways will be determined. Possible changes in the nature of trans-acting factors during development will be investigated by introducing SV40 RNA (or DNA) into X. laevis oocytes, ova and embryos. Finally, regulation of genes that encode splicing factors, the human U1 and U2 genes, will be studied, and the possibility that SV40 T antigen, or a cellular homologue, can affect expression of these genes will be investigated.